Gene regulation, a fundamental process in biology, is typically investigated at the level of transcription, yet there is a growing recognition of consequential post-transcriptional control of gene expression. In animals, microRNAs (miRNAs) and other small RNAs direct much of post-transcriptional regulation. MicroRNAs are regulatory RNAs that repress gene expression by interacting with messenger RNAs (mRNAs). In humans, miRNAs contribute to most biological pathways, and mutations perturbing miRNAs are implicated in disease. The unifying theme of all small RNA pathways is the silencing complex, which contains a small RNA and an Argonaute protein, the core proteins ofthe silencing complex. We have recently discovered two novel roles for an individual Argonaute protein, AG04, in male gametogenesis in mice. Ago4 is required for both the correct timing of entry into meiosis, a specialized form of cell division in the germline, and, for silencing ofthe sex chromosomes in meiosis, which is essential to the male germline. These are the first specific biological functions attributed to Ago4. Moreover, our data suggests that Ago3 shares some of these Ago4 functions. Because AGO proteins always function with small RNAs, our data directly implicate small RNAs in the regulation of meiotic entry and meiotic silencing. Our major goals for this proposal are to identify the small RNAs that participate in meiotic entry and meiotic silencing, and, to define their regulatory targets. Our first aim is to use high-throughput sequencing to identify the small RNAs and mRNAs expressed at different times of germline development in mice. To gain insights into gene regulation in the germline, we will also perform sequencing of mouse strains deficient in Ago3, Ago4, or both. In our second aim, we will use the sequencing data from aim I, together with computational approaches, to determine the mRNA targets of germline small RNAs. In our final aim, we will explore the biological significance of specific regulatory events in germline function and development using transgenic mice. Because meiotic entry and meiotic silencing are fundamental processes in germline biology, our studies will contribute to a better understanding of human reproductive biology and health.